Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[²H₄]-choline as a substrate. The limiting rate constants k_lim1 and k_lim2 at saturating substrate were well separated (k_lim1/k_lim2>9), and were >15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that k_lim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that k_lim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15°C and 39°C the k_lim1 and k_lim2 values increased with increasing temperature, allowing for the analyses of H⁺ and H^- transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the k_lim1 value (^H2Ok_lim1/^D2Ok_lim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the k_lim2 value gave lines with the slope_(choline)>slope_(D-choline), suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed.